Evaluation of immunogenicity and protective efficacy of the outer membrane porin F (OprF) against Pseudomonas plecoglossicida in large yellow croaker (Larimichthys crocea).

Large yellow croaker (Larimichthys crocea) farming dominates the marine aquaculture industry in China. However, the epidemic outbreaks of visceral white nodules disease (VWND), caused by bacterial pathogen Pseudomonas plecoglossicida, have emerged as a significant concern within the large yellow croaker industry. Although vaccination is considered to be an effective method for preventing and controlling P. plecoglossicida infection, there is currently no commercially available vaccine targeting this bacterium. In the present study, the outer membrane porin F (OprF) of P. plecoglossicida was characterized and revealed a high sequence similarity with that of other Pseudomonas species. The recombinant OprF protein (rOprF) produced in Escherichia coli was then evaluated for its immunogenicity and protective role against P. plecoglossicida in large yellow croaker. The rOprF was identified to have immunogenicity by Western blot using large yellow croaker anti-P. plecoglossicida sera. Additionally, the indirect immunofluorescence assay (IIFA) provided evidence indicating the surface exposure of OprF in P. plecoglossicida. Fish vaccinated twice via intraperitoneal (IP) injection with the purified rOprF combined with commercial adjuvant ISA 763A VG exhibited a relative percent survival (RPS) of 70.60% after challenge with virulent P. plecoglossicida strain through immersion. The administration of rOprF resulted in a notable increase in specific serum antibody levels and serum lysozyme activity compared to the control groups. The immune-related genes in the spleen and head kidney of rOprF-vaccinated fish were remarkably upregulated compared with the PBS-vaccinated sham group after the P. plecoglossicida challenge. In summary, the findings of this study suggest that rOprF exhibits considerable potential in inducing a robust immune response, making it a viable candidate for vaccination against P. plecoglossicida infection in large yellow croaker.

Characterization of inulin-type fructans from two species of Radix Codonopsis and their oxidative defense activation and prebiotic activities.

BACKGROUND: Codonopsis pilosula and C. tangshen are both plants widely used in traditional Chinese medicine. Polysaccharides, which are their primary active components, are thought to be important in their extensive use. In this study, two neutral polysaccharide fractions of C. pilosula (CPPN) and C. tangshen (CTPN) were obtained by fractionation on a DEAE-Sepharose column and characterized. RESULTS: It was confirmed that the neutral polymers CPPN and CTPN were ß-(2,1)-linked inulin-type fructans with non-reducing terminal glucose, and degree of polymerization (DP) of 19.6 and 25.2, respectively. The antioxidant and prebiotic activities in vitro were assayed based on IPEC-J2 cell lines and five strains of Lactobacillus. Results indicated that the effects of CPPN and CTPN were increased antioxidant defense in intestinal epithelial cells through enhanced cell viability, improved expression of total antioxidant capacity, glutathione peroxidase, superoxide dismutase and catalase, and reduced levels of malondialdehyde and lactic dehydrogenase. The prebiotic activity of CPPN and CTPN was demonstrated by the promoting effect on Lactobacillus proliferation in vitro. The different biological activities obtained between the two fractions are probably due to the different DP and thus molecular weights of CPPN and CTPN. CONCLUSION: The inulin fractions from C. pilosula and C. tangshen were natural sources of potential intestinal antioxidants as well as prebiotics, which will be valuable in further studies and new applications of inulin-containing health products. © 2020 Society of Chemical Industry.

Molecular characterization, expression and immune functions of two C-type lectin from Venerupis philippinarum.

In the present study, two C-type lectins (designated as VpClec-3 and VpClec-4) were identified and characterized from the manila clam Venerupis philippinarum. Multiple alignment and phylogenetic relationship analysis strongly suggested that VpClec-3 and VpClec-4 belong to the C-type lectin family. In nonstimulated clams, the VpClec-3 transcript was dominantly expressed in the hepatopancreas, while the VpClec-4 transcript was mainly expressed in gill tissues. Both VpClec-3 and VpClec-4 mRNA expression was significantly upregulated following Vibrio anguillarum challenge. Recombinant VpClec-4 (rVpClec-4) was shown to bind lipopolysaccharide (LPS) and glucan in vitro, whereas recombinant VpClec-3 (rVpClec-3) only bound to glucan. In addition, rVpClec-3 and rVpClec-4 displayed broad agglutination activities towards Vibrio harveyi, Vibrio splendidus and V. anguillarum, while no agglutination activities towards Enterobacter cloacae or Aeromonas hydrophila were observed in rVpClec-3. Moreover, hemocyte phagocytosis was significantly enhanced by rVpClec-3 and rVpClec-4. All the results showed that VpClecs function as pattern recognition receptors (PRRs) with distinct recognition spectra and are potentially involved in the innate immune responses of V. philippinarum.

The Polysaccharides from Codonopsis pilosula Modulates the Immunity and Intestinal Microbiota of Cyclophosphamide-Treated Immunosuppressed Mice.

Based on previous studies about microflora regulation and immunity enhancement activities of polysaccharides from Codonopsis pilosula Nannf. var. modesta (Nannf.) L. T. Shen (CPP), there is little study on intestinal mucosal immunity, which is a possible medium for contacting microflora and immunity. In the present study, the BALB/c mice were divided into five groups (eight mice in each group), including a normal group (Con), a model control group (Model), and model groups that were administered CPP (50, 100, 200 mg/kg/d) orally each day for seven days after intraperitoneal injection of 60 mg/kg BW/d cyclophosphamide (CP) for three days. CPP recovered the spleen index and restored the levels of IFN-γ, IL-2, IL-10, as well as serum IgG. In addition, it elevated ileum secretory immunoglobulin A (sIgA), the number of Lactobacillus and acetic acid content in cecum. These results indicated that CPP plays an important role in the protection against immunosuppression, especially mucosa immune damage, and the inhibition of pathogenic bacteria colonization, which could be considered a potential natural source of immunoregulator.

A Pectic Polysaccharide from Sijunzi Decoction Promotes the Antioxidant Defenses of SW480 Cells.

Sijunzi Decoction (SJZD) is a formula used for the treatment of spleen deficiency and gastrointestinal diseases in Traditional Chinese Medicine. Polysaccharides are reported to be the main components of SJZD responsible for its bio-functions. However, highly purified and clearly characterized polysaccharides from SJZD are not well described. Here we obtained a purified polysaccharide (SJZDP-II-I) from SJZD using ion exchange chromatography and gel filtration. Structure analysis by FT-IR and NMR identified SJZDP-II-I as a typical pectic polysaccharide with homogalacturonan and rhamnogalacturonan type I regions and arabinogalactan type I and II as side chains. In vitro studies indicated that SJZDP-II-I treatment could significantly enhance the total antioxidant capacity of SW480 cells, resulting from the promoted expressions of antioxidant enzymes and their master regulator PGC-1α, which would be valuable for further research and applications.

Purification and Partial Structural Characterization of a Complement Fixating Polysaccharide from Rhizomes of Ligusticum chuanxiong.

Rhizome of Ligusticum chuanxiong is an effective medical plant, which has been extensively applied for centuries in migraine and cardiovascular diseases treatment in China. Polysaccharides from this plant have been shown to have interesting bioactivities, but previous studies have only been performed on the neutral polysaccharides. In this study, LCP-I-I, a pectic polysaccharide fraction, was obtained from the 100 °C water extracts of L. chuangxiong rhizomes and purified by diethylaminethyl (DEAE) sepharose anion exchange chromatography and gel filtration. Monosaccharide analysis and linkage determination in addition to Fourier transform infrared (FT-IR) spectrometer and Nuclear magnetic resonance (NMR) spectrum, indicated that LCP-I-I is a typical pectic polysaccharide, with homo-galacturonan and rhamnogalacturonan type I regions and arabinogalactan type I and type II (AG-I/AG-II) side chains. LCP-I-I exhibited potent complement fixation activity, ICH50 of 26.3 ± 2.2 µg/mL, and thus has potential as a natural immunomodulator.

[Effect of Transplanting Time on Growth,Dry Matter Accumulation and Distribution,and Yield of Ligusticum chuanxiong].

Objective: To study the effect of transplanting time on growth, dry matter accumulation and distribution,and yield of Ligusticum chuanxiong,in order to provide theoretical and practical basis for the choice of cultivation methods. Methods: Ligusticum chuanxiong which planted in Pengshan were used as materials, and rearched in the field plot experiment. By the single factor randomized block design, the effects on growth development,dry matter accumulation and distribution of different periods of Ligusticum chuanxiong were analyzed. Results: Transplanting time has an important influence on the development of Ligusticum chuanxiong after lodging, and the lodging time was positively related to transplanting time; dry matter translocated to the underground part in January of the second year, and the dry matter translocated to the aerial parts in February, the dry matter secondary translocated to the underground part during March and April. Conclusion: Transplanting time affects the growth of Ligusticum chuanxiong,and affects the accumulation and distribution of every dry matter during the process of growth. Ligusticum chuanxiong transplant on August 31,and distribute more manure to improve the growth of aerial parts in the early stages,and spread additional manure to improve the growth of root stock at second year in shoot growth period,which can make a good harvest.

[Effect of P and K Fertilization on Four Iridoids in Axial and Lateral Root of Gentiana crassicaulis].

Objective: To study the effect of phosphorus( P) and potassium( K) fertilization on four iridoids in axial and lateral root of Gentiana crassicaulis. Methods: Field experiment was conducted based on phosphorus and potassium,and the content of four iridoids was analyzed in the axial and lateral root of Gentiana crassicaulis. Results: Despite of different proportion of phosphorus and potassium,the content of four iridoids in axial root was always higher than that in the lateral root, and under normal fertilizer condition, the content of axial and lateral root was 12. 36% and 9. 84%, respectively. On the condition of phosphorus and potassium fertilization, the content of gentiopicroside and loganic acid were higher than that of the swertiamarin and sweroside both in the axial and lateral root. And the difference of the two parts could be increased by high potassium with appropriate phosphorus application. Conclusion: The four iridoids were more likely to accumulate in the axial root of Gentiana crassicaulis, and phosphorus and potassium fertilization, only affected the different degrees on the content of ingredients among two parts. Thus, more attention should be paid to the use of phosphorus and potassium fertilization, and the difference of the active ingredient content in the axial and lateral root of Gentiana crassicaulis.

[The Effection Study of Transplanting Techniques for Yield and Quality of Gentiana crassicaulis].

Objective: To study the difference in yield and quality of Gentiana crassicaulis with different transplanting period and transplanting methods, and to determine the optimum transplanting technique of Gentiana crassicaulis in Ludian. Methods: The variation in fresh weight,dry weight,dry discount rate, length, diameter, branches,the content of gentiopicroside,loganin acid,alcohol-soluble extract, total ash were measured, and made a comprehensive evaluation of yield and quality by gray relational distance ideal comprehensive evaluation method. Results: There was a big difference in yield and quality of Gentiana crassicaulis with different transplanting time and transplanting methods. Gentiana crassicaulis were transplanted in March 10 with the density of 25 cm × 24 cm,and overburden 1 cm and flat transplanting had the best comprehensive evaluation of yield and quality. Conclusion: The transplanting time of Gentiana crassicaulis in Ludian should begin at the end of early March,and reduce overburden soil on the traditional transplanting methods.

[Correlation of Agronomic Character of Panax notoginseng in Guangxi with Soil Physical and Chemical Properties].

Objective: To study the effect of physicochemical characteristics of soil on Panax notoginseng agronomic character, in order to offer the theory for selecting an appropriate soil to cultivate Panax notoginseng, and to build the production bases. Methods: To test and analyze Panax notoginseng agronomic character, to analyze the form of layered soil particles, p H value and soil fertility. At the same time, the analysis of Panax notoginseng plant height, leaf number, the length of taproot, the width of taproot, stem diameter, the width of residual part of stem, rootlet number, root fresh and dry weight and drying rate were carried out. Results: Panax notoginseng agronomic character had a large difference. The length of roots, the width of roots and a large number of rootlets had a promoting effect on accumulating biological yield. Nevertheless, the numbers of branches had no obvious impact on accumulating biological yield. Most of soil texture were silty clay or light clay of silty clay. Physical clay and coarse silt could promote the growth of Panax notoginseng. Sand grain had significant effect on Panax notoginseng growth. A reasonable use of nitrogenous fertilizer had a prominent promoting effect on the width of the medicine's root. In fact, the use of phosphorus potassium also had a great impact on Panax notoginseng growth. Conclusion: Panax notoginseng should be planted on the light clay which are loose and can irrigate and drain conveniently and contains many silty. The heavy clay soil which are defective aeration and contain cosmid has a bad influence of the growth of Panax notoginseng. It will cause a decrease of the yield of medicinal materials. Using nitrogenous fertilizer aptly also is an effective way to improve the quality and yield of Panax notoginseng.

[Effects of Chemical Fertilizers and Organic Fertilizer on Yield of Ligusticum chuanxiong Rhizome].

OBJECTIVE: To study the effects of different N, P, K and organic fertilizer (OF) on yield of Ligusticum chuanxiong rhizome, in order to provide the theoretical foundation for the establishment of standardization cultivation techniques. METHODS: The field plot experiments used Ligusticum chuanxiong rhizome which planted in Pengshan as material, and were studied by the four factors and five levels with quadratic regression rotation-orthogonal combination design. According to the data obtained, a function model which could predict the fertilization and yield of Ligusticum chuanxiong rhizome accurately was established. RESULTS: The model analysis showed that the yields of Ligusticum chuanxiong rhizome were significantly influenced by the N, P, K and OF applications. Among these factors, the order of increase rates by the fertilizers was K > OF > N > P; The effect of interaction between N and K, N and OF, K and OF on the yield of Ligusticum chuanxiong rhizome were significantly different. High levels of N and P, N and organic fertilizer, K and organic fertilizer were conducive to improve the yield of Ligusticum chuanxiong rhizome. The results showed that the optimal fertilizer application rates of N was 148.20 - 172.28 kg/hm2, P was 511.92 - 599.40 kg/hm2, K was 249.70 - 282.37 kg/hm2, and OF was 940.00 - 1 104.00 kg/hm2. CONCLUSION: N, P, K and OF obviously affect the yield of Ligusticum chuanxiong rhizome. K and OF can significantly increase the yield of Ligusticum chuanxiong rhizome. Thus it is suggested that properly high mount of K and OF and appropriate increasing N are two favorable factors for cultivating Ligusticum chuanxiong.

[Fertilizer Effect on Yield and Quality of Gentiana crassicaulis].

OBJECTIVE: To find the best fertilizer formula of yield-quality-fertilizer effect, and to establish a new comprehensive evaluation-fertilizer effect method. METHODS: Grey related degree analysis and DTOPSIS method were combined to comprehensive evaluate the 11 indexes of 14 fertilizer formulas of nitrogen, phosphorus and potassium, in order to explore the best dosage and ratio. RESULTS: The fertilizer requirements of the factors which contribute to the yield and quality of Gentiana crassicaulis were different and the most appropriate rates of nitrogen, phosphorus and potassium fertilizer were 21.49~42.03 kg/667 m2, 49.53~65.75 kg/667 m2 and 26.34~ 42.97 kg/667 m2, respectively. CONCLUSION: The fertilizer formula in this study can be widely used in the standardized plantation of Gentiana crassicaulis.

[Correlation Analysis Between Physicochemical Properties of Rhizosphere Soil and Appearance Characters of Codonopsis pilosula Root in Jiuzhaigou County].

OBJECTIVE: To study the physicochemical properties of rhizosphere soil and their influence on appearance characters of semi-natural cultivated Codonopsis pilosula root. METHODS: The comprehensive methods by combining outdoor investigating with indoor analyzing were applied. Rhizosphere soil samples and plant materials were collected from 35 collection points, the physicochemical properties of soil and the appearance characters of plant materials were investigated. RESULTS: The physical property of rhizosphere soil under semi-natural cultivated condition was good, the soil texture was coarse powder of loam, slightly alkaline. The cultivated layer contained abundant nutrient components, the average organic matter content of 35 soil samples was 60.66 g/kg,and 2.99 g/kg for total nitrogen, 115.46 mg/g for available nitrogen, 0.8 g/kg for total phosphorus, 6.43 mg/kg for available phosphorus, 23.69 g/kg for total potassium and 124.48 mg/kg for available potassium, respectively. The result of correlation analysis indicated that the physicochemical properties of rhizosphere soil had significant influence on the appearance characters of Codonopsis pilosula root. CONCLUSION: The semi-natural cultivated model can maintain the structure of soil and hold nutrient components, it is conducive to the growth of Codonopsis pilosula and the standardized production base establishment. To increase the yield of Codonopsis pilosula root, it is recommended to increase the application of nitrogen and phosphorus fertilizer.

[Quadratic Orthogonal Rotation Combination Design on Alisma orientalis of Fertilization].

OBJECTIVE: To study the effects of combined N, P, K and micronutrient fertilizers on the yield of Alisma orientalis tuber, and to optimize the fertilizer application rate. METHODS: Four factors five levels quadratic orthogonal rotation combination design was used. A function was established on nitrogen, phosphor, potassium and microelement fertilizer application rate with the yield of Alisma orientalis tuber. RESULTS: The established mathematical model was of high reliability for prediction with quadratic regression equation of R2 = 0. 8980. The order of increasing Alisma orientalis tuber yield was nitrogen > micronutrient fertilizer > potassium > phosphor. The results of the frequency analysis showed that for the target yield over 8 250 kg/hm2 and the confidence interval of 95%, the optimal fertilizer application rates were as follows :nitrogen of 241. 45 - 283. 55 kg/hm2, phosphor of 81. 14 - 208. 44 kg/hm2, potassium of 95. 57 - 239. 42 kg/hm2, and zinc fertilizer of 14. 32 - 16. 18 kg/hm2, boron fertilizer of 18. 84 - 19. 86 kg/hm2, and molybdenum fertilizer of 0. 151 -0. 159 kg/hm2 in micronutrient fertilizer. CONCLUSION: Nitrogen is related to the growth of Alisma orientalis, potassium promotes tuber bulking, micronutrient fertilizer consisted of zinc, boron and molybdenum fertilizer promotes Alisma orientalis growth and the absorption of nitrogen, phosphor and potassium. Moderate application of nitrogen, phosphorus, potassium, zinc, boron and molybdenum fertilizer can promote Alisma orientalis tuber yield. The nitrogen has the best effect.

[Influences of Base Fertilizer Dosage and Root Size on Quality and Yield of Gentiana crassicaulis Root].

OBJECTIVE: In order to optimize the base fertilizer dosage and root size of Gentiana crassicaulis in Ludian,Yulong, Yunnan,the differences in yield and quality of Gentiana crassicaulis caused by different base fertilizer dosage and root size were investigated. METHODS: The variation in fresh weight, dry weight, drying rate, length, diameter, branches as well as the contents of gentiopicroside, loganin acid, alcohol-soluble extract and total ash were determined. The ideal distance grey relational degree comprehensive evaluation method was applied to make a comprehensive evaluation on the quality and yield of Gentiana crassicaulis. RESULTS: There were big differences in yield and quality of Gentiana crassicaulis among different base fertilizer dosage and root size. 2 000 kg/667 m2 of manure, 20 kg/667 m2 of superphosphate, 100 kg/667 m2 of plant ash and mix size of transplanting roots showed the best comprehensive quality and the highest yield. CONCLUSION: 2 000 kg/667 m2 of manure, 20 kg/667 m2 of superphosphate,100 kg/667 m2 of plant ash, high quality transplanting roots and stagger certain depth to transplant are highly recommended to produce high quality and high yield of Gentiana crassicaulis in Ludian, Yulong, Yunnan.

Gas chromatography-mass spectrometry analysis of essential oils from five parts of Chaihu (Radix Bupleuri Chinensis).

OBJECTIVE: To analyze the essential oils from flowers, leaves, stems, roots, and fruits of Chaihu (Radix Bupleuri Chinensis). METHODS: We extracted essential oils from different parts of Chaihu (Radix Bupleuri Chinensis) using a steam distillation method. The essential oils were analyzed by gas chromatography-mass spectrometry (GC-MS). Data were collected in full scan mode (m/z 60-600). Volatile components were identified based on their retention indices and by comparing their mass spectra with those in the National Institute of Standards and Technology 2005 database, assisted by tandem mass spectrometry information. The relative content of each constituent was determined by area normalization. RESULTS: We identified 111 components, of which 12 were common to all 5 parts, 30 were found only in roots, 14 were found only in flowers, 6 were found only in leaves, 4 were found only in stems, and 17 were found only in fruits. CONCLUSION: Our results show that the stems, flowers, leaves, and fruits of Chaihu (Radix Bupleuri Chinensis) contain a high concentration of essential oils, and that the exact composition of the essential oils differs among the plant parts. To develop new medicines and make full use of the Chaihu (Radix Bupleuri Chinensis) resource, it is important to characterize the essential oils from different parts of the plant. In future research, it will be important to determine the pharmacological effects of the various components and the essential oil mixtures.

[Effects of hormone treatment, light and temperatures on sprouting characteristics of Bupleurum chinense].

Seeds of Bupleurum chinense cultivar, Zhongchai No. 1, were sowed in plastic pots which used the arable layer soil as the nursery bed and putted in the artificial climate incubator at various temperatures (15, 20, 25, 15-25 degrees C) and light (8,12 h) to germinate, respectively. The lower constant temperature (15 degrees ) and the higher constant temperature (25 "C) were not conducive to the sprouting characteristics of B. chinese. While they were able to enhance root activity to some extent; The seeding growth of B. chinese was significantly better in the variable temperature than correspondence in the constant temperature, significantly. The emergence speed, emergence index, vigor index and root activity of Bupleurum were improved under the 12 h of light-time, but the germination rate was not improved. The sprouting of Bupleurum's seeds could be improved to some extent by soaking with hormone, such as gibberellin, cytokinin, salicylic acid. Gibberellin promoted seeds' sprouting and seedings's root activity of Bupleurum, while salicylic acid increased the root activity of seeding. There is a significant influence of light, temperatures and hormone treatment on the germination of Zhongchai No. 1 seeds, and all three are remarkably interacted; It is beneficial to promote seed germination by the temperature (20 + 5) degrees C, lighting (8 h) and gibberellin concentration (10 x 10(-6)).

[Optimum harvest study of Gentiana crassicaulis in Ludian].

The paper is aimed to study the difference in yield and quality at different harvest time and determine the optimum harvest of planting Gentiana in Ludian traditional harvest period. The authors analyzed the variation in fresh weight, dry weight, dry discount rate, length, diameter, volume and the content of gentiopicroside, loganin acid, alcohol-soluble extract and total ash and made a comprehensive appraisal of yield, appearance quality and intrinsic quality by gray relational distance ideal Comprehensive Evaluation method. The results showed that there is a big difference in yield and quality both 2-year-old and 3-year-old Gentiana harvested in traditional harvest period and the comprehensive evaluation more better when harvested more later. It can be seen, Gentiana harvested the later had a better yield and quality in Ludian traditional harvest period. The harvest of Gentiana can be appropriate delayed depending on the particular circumstances of production.

[Evaluation of Bupleuri Radix resources in Qingchuan based on DTOPSIS and grey related degree].

In order to select high quality and suitable Bupleuri Radix varieties in Qingchuan, and establish a new comprehensive method to evaluation the quality of Bupleuri Radix, 12 characters of 14 samples were evaluated by DTOPSIS and grey related degree. The results showed that varieties No. 8 and No. 10 had high quality. DTOPSIS and grey related degree gave the uniformity result, and the biggest difference of value of Ci in DTOPSIS method was 46. 33% , but the biggest difference of the weighting correlation number( r (i)) in grey related degree was only 13.10%. The DTOPSIS combined with grey related degree can evaluate the quality of Bupleuri Radix comprehensively and objectively.